1. Field of the Invention
The present invention relates to a viral mutant capable of selectively killing tumor cells. More particularly, the present invention relates to a viral mutant capable of selectively killing tumor cells by a combination of viral mediated oncolysis and anti-cancer (xe2x80x9csuicidexe2x80x9d) gene therapy.
2. Related Art
Neoplasia is a process that occurs in cancer, by which the normal controlling mechanisms that regulate cell growth and differentiation are impaired, resulting in progressive growth. This impairment of control mechanisms allows a tumor to enlarge and occupy spaces in vital areas of the body. If the tumor invades surrounding tissue and is transported to distant sites it will likely result in death of the individual.
In 1999, in the United States alone, approximately 563,100 people, or about 1,500 people per day, are expected to die of cancer. (Landis, et al., xe2x80x9cCancer Statistics, 1999, xe2x80x9d CA Canc. J. Clin. 49:8-31 (1999)). Moreover, cancer is a leading cause of death among children aged 1 to 14 years, second only to accidents. Id. Thus, clearly there is a need for the development of new cancer therapies.
1. Common Limitations of Conventional Therapies
The desired goal of cancer therapy is to kill cancer cells preferentially, without having a deleterious effect on normal cells. Several methods have been used in an attempt to reach this goal, including surgery, radiation therapy, and chemotherapy.
Surgery was the first cancer treatment available, and still plays a major role in diagnosis, staging, and treatment of cancer, and may be the primary treatment for early cancers (see Slapak, C. A., and Kufe, D. W., xe2x80x9cPrinciples of Cancer Therapy,xe2x80x9d in Harrison ""s Principles of Internal Medicine, Fauci, A. S. et al., eds., 14th Ed., McGraw-Hill Cos., Inc., New York, 1998, at 524). However, although surgery may be an effective way to cure tumors confined to a particular site, these tumors may not be curable by resection due to micrometastatic disease outside the tumor field. Id. Any cancer showing a level of metastasis effectively cannot be cured through surgery alone. Id.
Radiation therapy is another local (nonsystemic) form of treatment used for the control of localized cancers. Id. at 525. Many normal cells have a higher capacity for intercellular repair than neoplastic cells, rendering them less sensitive to radiation damage. Radiation therapy relies on this difference between neoplastic and normal cells in susceptibility to damage by radiation, and the ability of normal organs to continue to function well if they are only segmentally damaged. Id. Thus, the success of radiation therapy depends upon the sensitivity of tissue surrounding the tumor to radiation therapy. Id. Radiation therapy is associated with side effects that depend in part upon the site of administration, and include fatigue, local skin reactions, nausea and vomiting. Id. at 526. In addition, radiation therapy is mutagenic, carcinogenic and teratogenic, and may place the patient at risk of developing secondary tumors. Id.
Other types of local therapy have been explored, including local hyperthermia (Salcman et al., J Neuro-Oncol. 1:225-236 (1983)), photodynamic therapy (Cheng et al, Surg. Neurol. 25:423-435 (1986)), and interstitial radiation (Gutin et al., J. Neurosurgery 67:864-873 (1987)). Unfortunately, thus far these therapies have been met with only moderate success.
Local treatments, such as radiation therapy and surgery, offer a way of reducing the tumor mass in regions of the body that are accessible through surgical techniques or high doses of radiation therapy. However, more effective local therapies with fewer side effects are needed. Moreover, these treatments are not applicable to the destruction of widely disseminated or circulating tumor cells eventually found in most cancer patients. To combat the spread of tumor cells, systemic therapies are used.
One such systemic treatment is chemotherapy. Chemotherapy is the main treatment for disseminated, malignant cancers. (Slapak, C. A., and Kufe, D. W., xe2x80x9cPrinciples of Cancer Therapy,xe2x80x9d in Harrison ""s Principles of Internal Medicine, Fauci, A. S. et al., eds., 14th Ed., McGraw-Hill Cos., Inc., New York, 1998, 527). However, chemotherapeutic agents are limited in their effectiveness for treating many cancer types, including many common solid tumors. See id This failure is in part due to the intrinsic or acquired drug resistance of many tumor cells. See id. at 533. Another drawback to the use of chemotherapeutic agents is their severe side effects. See id. at 532. These include bone marrow suppression, nausea, vomiting, hair loss, and ulcerations in the mouth. Id. Clearly, new approaches are needed to enhance the efficiency with which a chemotherapeutic agent can kill malignant tumor cells, while at the same time avoiding systemic toxicity.
2. Challenges Presented by Central Nervous System Tumors Another problem presented in cancer treatment is that certain types of cancer, e.g., gliomas, which are the most common primary tumor arising in the human brain, defy the current modalities of treatment. Despite surgery, chemotherapy, and radiation therapy, glioblastoma multiforme, the most common of the gliomas, is almost universally fatal (Schoenberg, in Oncology of the Nervous System, M. D. Walker, ed., Boston, Mass., Martinus Nijhoff (1983); Levin et al., Chapter 46 in Cancer: Principles and Practice of Oncology, vol. 2, 3rd ed., De Vita et al., eds., Lippincott Press, Philadelphia (1989), pages 1557-1611).
Gliomas represent nearly 40% of all primary brain tumors, with glioblastoma multiforme constituting the most malignant form (Schoenberg, xe2x80x9cThe Epidemiology of Nervous System Tumors,xe2x80x9d in Oncology of the Nervous System, Walker, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1983)). The five year survival rate for persons with this high grade type of astrocytoma is less than 5 percent, given the current treatment modalities (surgery, radiation therapy and/or chemotherapy) (Mahaley et al., Neurosurgery 71: 826-836 (1989); Schoenberg, in Oncology of the Nervous System, Walker, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1983); Kim et al., J. Neurosurg. 74:27-37 (1991), Daumas-Duport et al., Cancer 2:2152-2165 (1988)). After treatment with radiation therapy, glioblastomas usually recur locally. Hochberg et al., Neurology 30: 907 (1980). Neurologic dysfunction and death in an individual with glioblastoma are due to the local growth of the tumor. Systemic metastases are rare. Id. For this reason, regional cancer therapy methods, rather than systemic methods, may be especially suitable for the treatment of glioblastomas.
Moreover, glioblastomas are resistant to many chemotherapeutic agents, perhaps due to the proliferative characteristics of this tumor type. Many chemotherapeutic agents are cell-cycle-active, i.e., cytotoxic primarily to actively cycling cells (Slapak, C. A., and Kufe, D. W., xe2x80x9cPrinciples of Cancer Therapy,xe2x80x9d in Harrison""s Principles of Internal Medicine, Fauci, A. S. et al., eds., 14th Ed., McGraw-Hill Cos., Inc., New York, 1998, 527). Generally, chemotherapy is most effective for cancers-with a small tumor burden where the growth fraction of the tumor is maximal. Id. The growth fraction for glioblastoma tumors is only 30%, with the remaining 70% of cells being in G0, a resting phase (cells in G0 may die or may re-enter the active cell cycle; Yoshii et al., J. Neurosurg. 65:659-663 (1986)). While the 30% of glioblastoma cells that are actively dividing contribute to the lethal progression of this tumor, the 70% that are quiescent are responsible for the resistance of these tumors to a number of chemotherapeutic agents that target actively proliferating cells.
Unfortunately, regional treatments, such as surgery and radiation therapy have also found limited success in the treatment of glioblastomas. (Burger et al., J. Neurosurg. 58:159-169 (1983); Wowra et al., Acta Neurochir. (Wien) 99:104-108 (1989); Zamorano et al., Acia Neurochir. Suppl. (Wien) 46:90-93 (1989)). Surgical treatment methods for glioblastomas are hampered by the lack of distinct boundaries between the tumor and the surrounding parenchyma, and by the migration of tumor cells in the white matter tracts extending out from the primary site (Burger et al., J. Neurosurg. 58:159-169 (1983)), which preclude their complete removal. Radiation therapy, which targets rapidly proliferating cells, is limited by the low growth fraction in glioblastomas, and by the radiation sensitivity of adjacent normal tissue (Wowra et al., Acta Neurochir. (Wien) 99:104-108 (1989); Zamorano et al., Acta Neurochir. Suppl. (Wien) 46:90-93 (1989)). Thus, new approaches are needed to treat brain tumors.
One non-traditional therapeutic method employs viruses to target neoplastic cells. Proposed viral cancer therapies include two distinct approaches: (i) direct cell killing (oncolysis) by a mutagenized virus (Martuza et al., Science 252:854-856 (1991); Mineta et al., Nature Med 1:938-943 (1995); Boviatsis et al., Cancer Res. 54: 5745-5751 (1994); Kesari, et al., Lab. Invest. 73: 636-648 (1995); Chambers et al., Proc. Natl. Acad. Sci. USA 92: 1411-1415 (1995); Lorence, R. M. et al., J. Natl. Cancer. Inst. 86: 1228-1233 (1994); Bischoff, et al., Science 274: 373-376 (i996); Rodriguez et al., Cancer Res. 57: 2559-2563 (1997)), and (ii) the use of viral vectors to deliver a transgene whose expression product activates a chemotherapeutic agent (Wei et al., Human Gene Therapy 5: 969-978(1994); Chen and Waxman, Cancer Res. 55: 581-589 (1995); Moolten, Cancer Gene Ther. 1: 279-287 (1994); Fakhrai et al., Proc. Natl. Acad. Sci. USA 93: 2909-2914 (1996); Roth et al., Nature Med. 2: 985-991 (1996); Moolten, Cancer Res. 46: 5276-5281 (1986); Chen et al., Proc. Natl. Acad. Sci. USA 91: 3054-3057 (1994)).
1. Viral Oncolysis
With regard to the first approach in viral cancer therapy, viral oncolysis, the genetic engineering of viruses for use as oncolytic agents has initially focused on the use of replication-incompetent viruses. This strategy was hoped to prevent damage to non-tumor cells by the viruses. A major limitation of this approach is that these replication-incompetent viruses require a helper virus to be able to integrate and/or replicate in a host cell. One example of the viral oncolysis approach, the use of replication-defective retroviruses for treating nervous system tumors, requires the implantation of a producer cell line to spread the virus. These retroviruses are limited in their effectiveness, because each replication-defective retrovirus particle can enter only a single cell and cannot productively infect others thereafter. Therefore, they cannot spread far from the producer cell, and are unable to completely penetrate a deep, multilayered tumor in vivo. Markert et al., Neurosurg. 77: 590 (1992) Ram et al., Nature Medicine 3:1354-1361 (1997).
More recently, genetic engineering of oncolytic viruses has focused on the generation of xe2x80x9creplication-conditionalxe2x80x9d viruses in an attempt to avoid systemic infection, while allowing the virus to spread to other tumor cells. Replication-conditional viruses are designed to preferentially replicate in actively dividing cells, such as tumor cells. Thus, these viruses should target tumor cells for oncolysis, and replicate in these cells so that the virus can spread to other tumor cells.
Some recent strategies for creating replication-conditional viral mutants as novel anticancer agents have employed mutations in selected adenoviral or herpes simplex virus type 1 (HSV-1) genes to render them viral replication-conditional (Martuza et al, Science 252:854-856 (1991); Mineta et al., Nature Med 1:938-943 (1995); Boviatsis et al., Cancer Res. 54: 5745-5751 (1994); Kesari, et al., Lab. Invest. 73: 636-648 (1995); Chambers et al., Proc. Natl. Acad Sci. USA 92: 1411-1415 (1995); Lorence, R. M. et al., J. Natl. Cancer. Inst. 86: 1228-1233 (1994); Bischoff, et al., Science 274: 373-376 (1996); Rodriguez et al., Cancer Res. 57: 2559-2563 (1997)). For example, an adenovirus with a deletion in the E1B-55Kd encoding gene has been shown to selectively replicate in p53-defective tumor cells (Bischoff, et al., Science 274: 373-376 (1996)). HSV-1 with deletions or insertions in viral genes encoding for thymidine kinase (Hstk) (Martuza et al., Science 252:854-856 (1991)), or ribonucleotide reductase (Hsrr) (Goldstein and Weller, J. Virol. 62: 196-205 (1988); Mineta et al., Gene Therapy 1:S78 (1994), Mineta et al., J. Neurosurg. 80:381 (1994); Mineta et al., Nature Med 1: 938-943 (1995); Boviatsis et al., Cancer Res. 54: 5745-5751 (1994)), or xcex334.5 (Mineta et al., Nature Med 1:938-943 (1995); Chambers et al., Proc. Natl. Acad. Sci. USA 92: 1411-1415 (1995)), have also been shown to replicate in and lyse dividing cells but not quiescent cells, presumably because the former can complement the defective viral function (Goldstein and Weller, J. Virol. 62: 196-205 (1988)).
However, these replication-conditional viral mutants have drawbacks. For example, the thymidine kinase deficient (TKxe2x88x92), viral mutant described by Martuza et al. (called dlsptk) (Science 252:854-856 (1991)), is only moderately attenuated for neurovirulence and produced encephalitis at the doses required to kill the tumor cells adequately (Markert et al., Neurosurgery 32:597 (1993)). Residual neurovirulence, as evidenced by a 50% lethality of intracranially-administered, replication-deficient herpes simplex virus viral vectors at 106 plaque forming units (pfu), limits the use of such vectors for tumor therapy. Furthermore, known TKxe2x88x92 HSV-1 mutants are insensitive to acyclovir and ganciclovir, the most commonly used and efficacious anti-herpetic agents, and thus undesired viral spread cannot be controlled using these drugs.
Moreover, the HSV-1 RRxe2x88x92 mutant with insertion of an Escherichia coli lacZ gene into the large subunit (ICP6) of Hsrr described by Goldstein and Weller, J. Virol. 62: 196-205 (1988), may be susceptible to spontaneous regeneration of the wild-type viral gene, which would render the virus replication competent in normal cells. An alternative ICP6 HSV-1 mutant, which is described in U.S. Pat. No. 5,585,096, was designed to contain a deletion mutation in the xcex334.5 gene in addition to the insertion of lacZ into ICP6, because the chance of reversion to the wild-type gene is smaller for a large deletional or substitutional mutation than for an insertional mutation. However, the oncolytic effect of both of these RR mutants, and other replication-conditional mutants that require cellular complementation of some factor for replication, is limited by tumor cell heterogeneity (Sidranski et al., 355: 846-847 (1992); Bigner et al, J. Neuropathol. Exp. Neurol. 40: 201-229 (1981)) for the cellular factor(s) necessary to complement the deficiencies of the viral mutant. Moreover, the viral oncolysis based approaches discussed above are limited by antiviral immune responses, as well as the possibility of host fever interfering with viral replication (for temperature sensitive mutants).
2. Viral Delivery of Anticancer Transgenes
As mentioned above, the second approach in viral cancer therapy is the viral delivery of anticancer transgenes (Wei et al., Human Gene Therapy 5: 969-978 (1994); Chen and Waxman, Cancer Res. 55: 581-589 (1995); Moolten, Cancer Gene Ther. 1: 279-287 (1994); Fakhrai et al., Proc. Natl. Acad. Sci. USA 93: 2909-2914 (1996); Roth et al., Nature Med. 2: 985-991 (1996); Moolten, Cancer Res. 46: 5276-5281 (1986); Chen et al., Proc. Natl. Acad. Sci. USA 91: 3054-3057 (1994); Mroz, and Moolten, Hum. Gene Ther. 4: 589-595 (1993); Mullen et al., Proc. Natl. Acad. Sci. USA 59: 33-37 (1992); Wei et al., Clin. Cancer Res. 1: 1171-1177 (1995); Marais et al., Cancer Res. 56: 4735-4742 (1996); Chen et al, Cancer Res. 56: 1331-1340 (1996)). It has been proposed that genes with a drug-conditional xe2x80x9ckillingxe2x80x9d function (also referred to as suicide genes) be employed for treating tumors.
In one example of viral delivery of a suicide gene, expression of the HSV thymidine kinase (Hstk) gene in proliferating cells, was found to render cells sensitive to the deoxynucleoside analog, ganciclovir (GCV) (Moolten et al, Cancer Res. 46:5276-5281 (1986); Moolten et al., Hum. Gene Ther. 1:125-134 (1990); Moolten et al., J. Natl. Cancer Inst. 82:297-300 (1990)). HSV-TK mediates the phosphorylation of GCV, which is incorporated into DNA strands during DNA replication (S-phase) in the cell cycle, leading to chain termination and cell death (Elion, G. B., J. Antimicr. Chemother. 12, sup. B:9-17 (1983)). Cells bearing a retroviral vector carrying HSV-TK and implanted into brain tumors growing in human patients have been demonstrated to confer sensitivity to the anti-herpes drug GCV (Oldfield et al., Hum. Gene Ther. 4:39 (1993)). Of eight patients with recurrent glioblastoma multiforme or metastatic tumors treated by stereotactic implantation of murine fibroblast cells producing these retroviral vectors, five patients demonstrated some evidence of anti-tumor efficacy but none were cured (Culver, Clin. Chem 40: 510 (1994)).
These retroviral vectors are replication-incompetent, therefore viral spread is dependent on the implantation of a producer cell line. Thus, this type of viral therapy is subject to the following limitations: (1) low viral titer; (2) limitation of viral spread to the region surrounding the producer cell implant; (3) possible immune response to the producer cell line; (4).possible insertional mutagenesis and transformation of retroviral infected cells; (5) single treatment regimen of the pro-drug, GCV, because the xe2x80x9csuicidexe2x80x9d product kills retrovirally infected cells and producer cells; and (6) limitation of the bystander effect to cells in direct contact with retrovirally transformed cells (Bi et al., Human Gene Therapy 4: 725 (1993)). Oldfield et al. (1993), supra. In addition, for therapies using drugs such as GCV, the dependence on the occurrence of DNA replication during drug exposure may limit its therapeutic effectiveness. For instance, because the majority of cells in human malignant brain tumors are in G0 (resting phase) at any one time (Nagashima et al., Acta Neuropathol. 66:12-17 (1985); Yoshii et al., J. Neurosurg. 65:659-663 (1986)), the majority of cells would not be targeted by transient exposure to the drug.
Another example of a suicide gene suitable for viral delivery is the cytochrome P450 gene, which confers chemosensitivity to the class of oxazaphosphorine drugs. Two of these drugs, cyclophosphamide (CPA) and its isomeric analog ifosfamide (IFA) are mainstays of cancer chemotherapy for several types of tumors (Colvin, O. M., in Cancer Medicine, Holland et al., eds., Lea and Febiger, Philadelphia, Pa. (1993), pages 733-734). These therapeutically inactive prodrugs require bioactivation by liver-specific enzymes of the cytochrome P450 family. One of these enzymes, cytochrome P450 2B1 (xe2x80x9cCYP2B1xe2x80x9d), which is induced by phenobarbital, activates CPA and IFA with high efficiency (Clarke et al., Cancer Res. 49:2344-2350 (1989); Weber and Waxman, Biochem. Pharm. 45:1685-1694 (1993)). CPA and IFA are hydroxylated by cytochrome P450 to yield the primary metabolites, 4-hydroxycyclophosphamide or 4-hydroxyifosphamide, respectively. These primary metabolites are unstable and spontaneously decompose into cytotoxic compounds: acrolein and phosphoramide (or ifosphoramide) mustard (Colvin et al., Cancer Treat. Rep. 65:89-95 (1981); Sladek, in Metabolism and Action of Anticancer Drugs, Powis et al., eds., Taylor and Francis, New York (1987), pages 48-90). The latter causes interstrand cross-links in DNA regardless of cell-cycle phase. Maximum cytotoxicity is obtained during subsequent synthesis (S) and mitotic (M)-phases of the cell cycle due to strand breaks (Colvin (1993), supra). U.S. Pat. No. 5,688,773, to Chiocca et al. (Nov. 18, 1997), describes a gene therapy paradigm using cytochrome P450 and CPA.
The inventors and others have employed replication-defective vectors based on retrovirus (Wei et al., Human Gene Therapy 5: 969-978 (1994); Chiocca et al., U.S. Pat. No. 5,688,773), or adenovirus (Chen et al., Cancer Res. 56: 1331-1340 (1996)) to achieve transfer into tumor cells of the transgene encoding rat CYP2B1. When treated with CPA, tumor cells engineered to express cytochrome CYP2B1 generate freely diffusible active CPA metabolites that are cytotoxic to surrounding tumor cells, which may not contain the CYP2B1 transgene (Chen and Waxman, Cancer Res. 55: 581-589 (1995); Wei et al., Clin. Cancer Res. 1: 1171-1177 (1995)). Thus, the CPA/cytochrome P450 gene therapy approach may provide a means for intratumoral generation of alkylating metabolite.
Without the expression of P450 to provide local activation, conversion of oxazaphosphorine anti-cancer drugs, such as CPA, into their active metabolites is primarily restricted to the liver. Thus, typically, the active metabolites, are distributed systemically. Due to the toxicity of the active metabolites, oxazaphosphorine drugs may not be able to be administered at sufficiently high levels to effectively kill the tumor, without also causing systemic toxicity in the patient, and possibly death. Moreover, oxazaphosphorines are largely ineffective in treating tumors of the central nervous system (CNS) owing to the poor transport of the activated metabolites across the blood-brain barrier and into cells (Genka et al., Cancer Chemother. Pharmacol. 27:1-7 (1990)), and to the low levels of cytochrome P450 found in brain and tumor cells (Hodgson et al., Mol. Cell. Biochem. 120:171-179(1993)).
Thus, one benefit of the CPA/cytochrome P450 gene therapy approach is the intratumoral generation of alkylating metabolite. By providing elevated concentrations of the anticancer agent in the tumor, this approach may reduce the exposure of normal cells to toxic metabolites and thus reduce the amount of drug required to be administered. However, none of the gene based approaches presently available, including the CPA/CYP2B1 paradigm described above, offer the benefit of combined viral mediated oncolysis with suicide gene mediated oncolysis.
While both the virus-based and the gene-based approaches have provided evidence of significant therapeutic effects in animal models of tumors, each method suffers from inherent limitations. Although the virus-based approach theoretically provides the potential for extensive replication of the virus with spread in the tumor mass, its effects are limited by the efficiency of viral infection; the requirement of a helper virus or producer cell line for some viral vectors; tumor cell heterogeneity (Sidranski et al., 355: 846-847 (1992); Bigner et al, J. Neuropathol. Exp. Neurol. 40: 201-229 (1981)) for the cellular factor(s) complementing viral mutant growth for other viral vectors; and antiviral immune responses.
In the gene-based approaches tested thus far, the efficiency of transduction of cells within a tumor mass is limited by the defective nature of the vector. In fact, the majority of positively transduced cells occurs within a few cell layers from the site of vector inoculation (Nilaver et al. Proc. Natl. Acad. Sci. USA 21: 9829-9833 (1995); Muldoon et al., Am. J. Pathol. 147: 1840-1851 (1995); Ram Z. et al., J. Neurosurg. 82, 343A (abst.)(1995)). Moreover, even for viral vector systems where a producer cell line is unnecessary, or not killed by the suicide gene/drug combination, viral replication may be inhibited by the drug used. Furthermore, where the suicide-gene/drug combination is TK/GCV, the ability of the drug to kill tumor cells is limited by the stage of the cell cycle of the cells as GCV targets only cells in the process of DNA replication. It is thus unlikely that therapeutic gene delivery by these replication-defective vectors will affect tumor cells distant from the inoculation site, even in instances where the therapeutic gene produces a freely diffusible anticancer agent, such as cytokines or CPA metabolites.
Therefore, it remains of utmost importance to develop a safe and effective viral mutant for selectively killing neoplastic cells. Although various attempts have been made to engineer a viral mutant able to kill human tumor cells in vivo, presently no viral mutant combines the benefits of both viral and gene-therapy based approaches, thereby compensating for the limitations of each. There exists a need for a viral mutant that can both target neoplastic cells for viral mediated oncolysis and deliver a transgene capable of activating or enhancing a chemotherapeutic agent locally, wherein the transgene/chemotherapeutic agent combination does not significantly inhibit viral replication.
Accordingly, the present invention overcomes the disadvantages of the prior art by providing a viral mutant that can selectively target neoplastic cells for viral oncolysis and deliver a transgene encoding a product capable of activating or enhancing a chemotherapeutic agent, a method of using this viral mutant and a pharmaceutical composition containing this viral mutant.
In a preferred embodiment of the invention, the viral mutant comprises a (a) a mutation in a viral gene whose mammalian homologue is up-regulated in cells with elevated levels of E2F; and (b) an insertion into this viral gene, of a transgene encoding a gene product capable of converting a chemotherapeutic agent to its cytotoxic form, where the chemotherapeutic agent does not significantly inhibit replication of the viral mutant.
The invention also provides an embodiment of the foregoing viral mutant, wherein the viral mutant is derived from a herpes virus, particularly where the herpes virus is a herpes simplex virus, and more particularly, where it is herpes simplex virus type 1 or type 2.
In another embodiment, the viral gene, whose mammalian homologue is up-regulated in cells with elevated levels of E2F, encodes ribonucleotide reductase (RR), or more particularly the large subunit of RR. In an even more preferred embodiment, this viral gene encodes ICP6. Alternatively, the gene encoding RR encodes the small subunit.
In addition, the invention provides an embodiment of the foregoing viral mutant where the transgene encodes cytochrome P450. More particularly, this cytochrome P450 may be P450 2B 1, or alternatively P450 2B6, P450 2A6, P450 2C6, P450 2C8, P450 2C9, P450 2C11, or P450 3A4.
The invention also provides an embodiment of the foregoing viral mutant, and wherein the chemotherapeutic agent is a member of the oxazaphosphorine class, and particularly, where this agent is cyclophosphamide, or alternatively is ifosfamide, N-methyl cyclophosphamide, methylchloropropylnitrosourea, polymeric cyclophosphamide, polymeric ifosfamide, polymeric N-methyl cyclophosphamide, or polymeric methylchloropropylnitrosourea.
In a preferred embodiment of the invention, the viral mutant is derived from a herpes virus, and comprises: (a) a mutation in a gene encoding ribonucleotide reductase; and (b) an insertion into said gene, of a transgene encoding a cytochrome P450. In a particularly preferred embodiment of the invention, the viral mutant is derived from HSV-1, and the mutation comprises a deletion in the large subunit of the ribonucleotide reductase gene, especially in ICP6, and the cytochrome P450 encoded is P450 2B1. Alternatively, the cytochrome P450 encoded is P450 2B6, P450 2A6, P450 2C6, P450 2C8, P450 2C9, P450 2C11, or P450 3A4. In a particularly preferred embodiment of the foregoing viral mutant, the viral mutant is rRp450.
The present invention also provides a method for selectively killing neoplastic cells, using the viral mutant described above, comprising the steps of: (a) infecting the neoplastic cells with a viral mutant comprising: (i) a mutation in a viral gene whose mammalian homologue is up-regulated in cells with elevated levels of E2F, and (ii) inserted into said viral gene, a transgene encoding a gene product capable of converting a chemotherapeutic agent to its cytotoxic form, wherein said chemotherapeutic agent does not significantly inhibit replication of said viral mutant; (b) contacting the neoplastic cells with the chemotherapeutic agent; and (c) selectively killing the neoplastic cells.
In addition, the invention provides a method for selectively killing neoplastic cells comprising the steps of: (a) infecting neoplastic cells with a viral mutant comprising: (i) a mutation in a gene encoding ribonucleotide reductase; and (ii) an insertion into this gene, of a transgene encoding a cytochrome P450; (b) contacting the neoplastic cells with a chemotherapeutic agent capable of being activated by the cytochrome P450; and (c) selectively killing the neoplastic cells. In a particularly preferred embodiment of this method, the viral mutant is derived from HSV-1, the mutation comprises a deletion in the large subunit of the ribonucleotide reductase gene, especially in ICP6, and the cytochrome P450 encoded is P450 2B 1. Alternatively, the cytochrome P450 encoded is P450 2B6, P450 2A6, P450 2C6, P450 2C8, P450 2C9, P450 2C11, or P450 3A4. In addition, the chemotherapeutic agent is preferably a member of the oxazaphosphorine class, particularly cyclophosphamide, ifosfamide, N-methyl cyclophosphamide, methylchloropropylnitrosourea, polymeric cyclophosphamide, polymeric ifosfamide, polymeric N-methyl cyclophosphamide, or polymeric methylchloropropylnitrosourea. In a particularly preferred embodiment of this method, the viral mutant is rRp450.
Another embodiment of the invention is a pharmaceutical composition containing the foregoing viral mutant, wherein this composition may also contain one or more pharmaceutically acceptable excipients.
Thus, the inventors have discovered that the combination of viral mediated oncolysis with activation, by the product of a transgene carried by the viral mutant, of a chemotherapeutic agent into metabolites that possess antineoplastic, but not antiviral-replication activity, provides a potentiated oncolytic effect much greater than that provided by either viral mediated oncolysis, or suicide gene therapy alone.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.